Issue No.29 / March 2010
MICROBIOLOGICAL TOOLS FOR QUALITY
ASSURANCE IN HATCHERY:
Laboratory Methods
By Dr Vincent TURBLIN, Deputy Regional Market Manager – Poultry - CEVA Animal Health Asia Pacific
Most of the time, the monitoring analysis are focusing on bacteria (e.g.
coliforms, Pseudomonas and Staphylococcus) and molds (including
Aspergillus). Virus isolation and/or PCR are indeed much more complicated to
implement routinely, and costly as well. And somehow, the bacterial and fungi
presence by itself will assess the efficacy of the sanitation procedures applied.
There are different kinds of procedure, depending upon the target to be
sampled: air, flat surfaces, eggshell, liquids, fluff or chicks. And, according to
the procedure, different material and media will be used for growing the germs
monitored.
Figure 1: Enterobacteriaceae
(Transmission Electron Microscopy)
MATERIAL REQUIRED FOR THE MICROBIOLOGICAL MONITORING
The samples will be taken thanks to material chosen, according to the procedure decided by the laboratory. For
growing the molds or bacteria, different kind of media can be used:
either agar, a solid medium whose hard jello consistency allows a direct sampling and then, incubation
on the same material
or broth, which is a liquid medium that will be preferred for isolating and identifying the specific
organisms which grow on agar media.
The agar media are generally used to determine the type and the number of microorganisms present. In order
to make comparisons of colony counts over time, it is important to use always the same kind of agar. Agar
media provides the nutrients required by microbes to grow. The various types of microbes have different nutrient
requirements, and their susceptibility to inhibitory compounds allow one to use a selective type of agar to
determine number and type of microbes present in a given location.
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WHICH MEDIUM SHOULD BE USED?
Choosing the type of agar will be done according to the purpose of the monitoring:
for troubleshooting problems and helping in identification of specific types of bacteria, specific media
should be used. Media for the detection of specific groups of organisms such as Pseudomonas,
Staphylococcus, Streptococcus and Aspergillus are commercially available as prepoured plates from a
number of scientific supply houses.
but for a routine monitoring of the total microbial load in the hatchery, as a
follow up of the sanitation and biosecurity procedure efficiency, three types
of media are particularly interesting: TSA for total bacteria, PDA for molds /
yeasts and Mac Conkey for coliforms.
Figure 2: E. coli (Mac Conkey)
First, general purpose media for bacteria like Tryptic soy agar (TSA) or nutrient agar don’t contain any
inhibitory chemical compounds and allow many types of bacteria to grow. However, TSA may be the better of
the two since it contains two peptones and is designed to support the growth of a wide variety of delicate or
fastidious organisms. Non-selective media are usually incubated at 37oC (98.6oF) for 48 hours before counting.
As regards Mac Conkey agar (MA), it is commonly used to detect coliform bacteria. Violet red bile agar (VRB)
and eosin methylen blue agar (EMB) can also be used, but Mac Conkey probably detects a slightly wider array
of bacteria than the other two. Mac Conkey agar is usually incubated at 37oC for 48 hours. Coliforms (including
E. coli) produce colonies which are red in color on MA, but clear or colorless should also be counted since these
colonies indicate the presence of other harmful bacteria (e.g. Salmonella).
Finally, Sabouraud dextrose agar or potato dextrose agar are general purpose media for molds and yeasts.
Potato dextrose agar (PDA) may be slightly superior to Sabouraud dextrose agar (SDA) for growing molds since
SDA was originally formulated to detect fungi associated with skin infection (such as ring worm). Media which
are used to enumerate molds will often include antibiotic(s) (such as chloramphenicol 0.05g/l) to inhibit bacterial
growth and allow the molds present to grow. To prevent loss of activity, antibiotics are generally added following
autoclaving and cooling of the medium. Molds tend to grow at a slower rate than bacteria. Thus, general
purpose mold media are generally incubated at room temperature for 3 to 5 days.
Figure 3: Aspergillus on eggs (left) and on Potatoe Dextrose Agar (right)
While pre-poured media are more expensive than dehydrated media prepared by your personnel at your facility,
the quality and sterility of these media are generally assured by the companies which prepare them. Since
media quality and sterility can affect the count obtained, quality and cost should be taken into consideration
when making the decision to purchase or prepare your own media.
CEVA ANIMAL HEALTH ASIA PACIFIC
3.06 Level 3, Wisma Academy – 4A, Jln 19/1 – 46300 P.Jaya – Selangor – Malaysia – Tel: +603 7957 4440 – Fax: +603 7954 4702
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HOW TO MAKE MEDIA FOR HATCHERY MONITORING?
First, the place where they should be prepared is very important: at least a room
within the hatchery which has minimal traffic flow and minimal airborne
contamination. The vaccine room might be a good media preparation room for
instance.
Then, tempered glass containers (such as pyrex) should be
used, according to the directions on the media package as a
guide. The amount of water to be mixed should be appropriate
and heated using a stirring hot plate. Prior to autoclaving,
media should be completely melted and dissolved, and they are so when they are clear not
cloudy. But caution should be taken not to overheat after clearing. The final media should
then be autoclaved at 15 psi of steam (121oC or 250oF) for 15 minutes or as indicated in the
package directions.
Caution: in case of too long autoclaving, sugars in the media may caramelize and peptones
may degrade like other heat sensitive media components. Thus overheated media should be
discarded. Since media are extremely hot following autoclaving, they should be allowed to
cool to the point where it can be handled with bare hands (approximately 110oF or 115oF).
Figure 4: Tempered
glass container for
media preparation
Regarding RODAC procedure (Replicate Organism Direct Agar Contact), Petri
dishes (15*100 mm) to be filled with media should be placed on a level counter
top or table. The plastic sleeves which contained new dishes should be saved for
plate storage after media is dispensed. Each plate will be filled with 16.5 to 17.5
mL (½ to 2/3 full). A strictly aseptic technique must be observed when pouring
plates. In order to avoid any spilling or bubble formation, the plates should be
handled carefully prior to solidification (however, to save space, plates may be
stacked as you pour them). The objective when pouring RODAC plates is to
make a perfectly convex surface with the agar. Thus, a level surface is important.
Figure 5: Convex surface on
Contact Plate (RODAC procedure)
After plates have cooled to room temperature, they should be incubated overnight at 37oC to check for sterility.
Plates which have colonies on them following incubation should be discarded, while those without colonies
should be stored in the plastic sleeves that were saved from new plates. Sleeves should be dated, inverted to
prevent condensation from falling on the media surface and placed in a refrigerator to minimize drying.
Should you decide to prepare your own agar media, equipment (hot plates, autoclaves and gram scale) and
supplies (such as glassware and plates) may be purchased from scientific supply houses.
CEVA ANIMAL HEALTH ASIA PACIFIC
3.06 Level 3, Wisma Academy – 4A, Jln 19/1 – 46300 P.Jaya – Selangor – Malaysia – Tel: +603 7957 4440 – Fax: +603 7954 4702
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MICROBIOLOGICAL PROCEDURE
INCUBATION OF PLATES:
Common rules of microbiology are obviously to be applied. In order to avoid
condensation to influence the growth, all plates should be incubated with the media
side of the plates on top.
For RODAC procedure, the incubation of large numbers of contact plates can be
organized by different ways, like the use of specific tracks.
Figure 6: RODAC tracks
For bacterial monitoring, a microbiological incubator or a setter (if a microbiological incubator is not
available) can be used. Indeed, the temperature of incubation should be about 37oC (98.6F) and the
time about 48 H. But if plates have to be incubated in a setter, the workers should be careful not to
disturb them. For this purpose, it is possible to protect them by a plastic bag (non fully closed
nevertheless).
As regards the mold monitoring, the incubation should be performed at room temperature for 3 to 5
days.
IDENTIFICATION OF MICROBIOLOGICAL GROWTH:
In general, bacterial colonies will be smooth, mucoid, and 1-3
mm diameter.
Mold colonies will be filamentous in appearance and may be
much larger due to the extended incubation period.
Figure 7: Escherichia coli on Mac Conkey Agar
Colonies on air plates, contact plates, liquid
spread plates, and swab streaks should be
counted and recorded. If a dilution had been
used before on the plate, a correction by the
dilution factor (10, 100, 1000, etc) has to be
brought obviously.
Figure 9: Counting after incubation
(liquid sample).
Figure 8: Counting after incubation
(surface/contact plate).
CEVA ANIMAL HEALTH ASIA PACIFIC
3.06 Level 3, Wisma Academy – 4A, Jln 19/1 – 46300 P.Jaya – Selangor – Malaysia – Tel: +603 7957 4440 – Fax: +603 7954 4702
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CONCLUSION:
Evaluating the microbial monitoring program is the most important step. It is necessary to make sure that all
results will be recorded and be able to notify any changes occurring over time. Consequently, the sampling
procedure must be well planned prior to its implementation and all the steps respected and recorded with
relevant identification of the samples. Only then, the results in each monitored area should be compared with
hatchability and chick livability data.
In this issue, the material necessary and the laboratory procedures of this microbiological monitoring have been
discussed. Depending on the hygiene and the structure of the monitored hatchery, targets can be posed in a
progression way to achieve this reference after a while. Excessive colonies will indicate poor sanitation
procedures or a hatching egg production problem.
It is well known that an early detection of contamination can minimize hatchery and chick quality problems,
which are of utmost importance for the broiler performance at the end of the day.
CEVA ANIMAL HEALTH ASIA PACIFIC
3.06 Level 3, Wisma Academy – 4A, Jln 19/1 – 46300 P.Jaya – Selangor – Malaysia – Tel: +603 7957 4440 – Fax: +603 7954 4702